Neuroscience and Behavioral Physiology, Vol. 34, No. 4, 2004

Comparative Morphological Analysis of Neuron Populations in the Ganglion Layer of the Rodent Cerebellum T. Ya. Orlyanskaya and T. M. Lyutikova

UDC 611.817.1.018:599.323.4

Translated from Morfologiya, Vol. 122, No. 5, pp. 50–53, September–October, 2002. Original article submitted October 3, 2001, revised version received January 13, 2002.

The basic principles of the neuronal organization of the vertebrate cerebellum have been described in a large number of studies, which indicate that the common structural plan of the cerebellum is constant for all mammals. However, at the level of lower taxonomic units, this part of the brain in mammals has acquired, during the course of evolutionary transformation, differences not only in terms of shape, size, and position, but also histological differentiation. Data have been obtained relating to individual variability in the neuronal organization of the cerebellum in terms of many criteria, both within taxa and phylogenetically diverse parts of the cerebellum. Purkinje cells (PC) in the ganglion layer have been shown to differ in terms of acetylcholinesterase activity [4, 8, 14], the state of the protein background [10], and the degree of chromophilia (“dark” and “light” cells are distinguished) [12]; there are variations in the ratio of hypo-, normo-, and hyperchromic cells within the PC population in members of a single class, order, and family, and there are variations in the number of connections with granule cells in members of different mammalian orders [1, 13]. Despite the existence of many investigations on the structural-functional organization of the cerebellum, many questions still remain unresolved. This applies to the quantitative characteristics of neuron populations in cerebellar cortical structures, so there is the possibility that new criteria for discriminating neurons and assessing the processes whereby animals’ adaptive responses appear at the level of cell populations will be identified. A number of authors [5, 7, 11, 13] link the characteristic structural organization of the brain and its compart-

ments with the ecological features in which species live and, above all, with the specific modus vivendi, means of feeding, and intensity of movement activity. From these points of view, comparative morphological study of the cerebella of the rodents, a mammalian order characterized by a high level of plasticity in motor functions and occupying all possible ecological niches and being directly relevant to human life, is of undoubted interest and allows us to identify and assess the characteristics of adaptive reactions of rodents of different ecological-morphological groups at the population-cellular level. The aim of the present work was to obtain a quantitative assessment of neuron populations in the ganglion layer of the cerebellar cortex of members of an ecological-morphological group of small burrowing animals.

MATERIALS AND METHODS Studies were performed on mature animals: 12 gray rats (Rattus norvegicus) weighing 272 ± 14 g, eight mole lemmings (Ellobius talpinus P.) weighing 40.0 ± 2.0 g, ten common voles (Microtus arvalis P.) weighing 25.0 ± 2.0 g), and eight house mice (Mus musculus) weighing 23.0 ± 0.8 g. Animals were decapitated under air-ether anesthesia. Cerebella were fixed in Carnaud’s fluid and embedded in paraffin. Frontal sections of thickness 5–7 µm were stained using a qualitative reaction for ribonucleoprotein complexes (RNP) with thionine as described by Nissl and modified by Viktorov [2]. Errors in studying and identifying conditions were reduced by checking section thickness (5–7 µm) and cover slips (0.14–0.15 mm) and using Apata’s fluid; reactions for RNP were performed on cerebellar sections from all groups of animals on a single slide [3]. Rodent ganglion layer neurons, i.e., cerebellar cortex PC, were

Department of Biology (Director: Professor T. M. Lyutikova), Omsk Medical Academy.

333 0097-0549/04/3404-0333 ©2004 Plenum Publishing Corporation

334

Orlyanskaya and Lyutikova

a

b

c

d

Fig. 1. Purkinje cells in the cerebella of burrowing rodents. a) House mouse (Mus musculus); b) mole lemming (Ellobius talpinus); c) common vole (Microtus arvalis); d) gray rat (Rattus norvegicus). Stained by Nissl thionine method. Magnification: objective ×40, ocular ×10.

studied. The numbers of cells with different degrees of chromophilia were counted, i.e., normo-, hypo-, and hyperchromic cells without structural alterations, along with total hyperchromic neurons, pyknotic neurons, and neurons with signs of marked chromatolysis, using a method described previously by Lyutikova [6]. The first three cell types were regarded as normal variants resulting from the relative stability of biological processes at the level of cell populations. Considering the specific characteristics of the distribution of neurons in the cerebellar cortex ganglion layer, the density of PC distribution in the upper part of the gyrus was determined using an MOV-1×15× ocular micrometer. At least 10 microscope fields were counted in each individual, and the variability of this measure was assessed for each species; mean PC distribution densities per mm of gyrus length were compared for all groups of animals studied.

Computerized morphometry based on Video-TestMorph program was used to measure the linear size of the cerebellar neurons of interest: body cross-sectional area (Sb), nuclear cross-sectional area (Sn), and cytoplasm crosssectional area (Sc), as well as the structural nuclear-cytoplasmic coefficient (sNCC). The statistical significance of the data was analyzed using Student’s t test.

RESULTS AND DISCUSSION The diversity of motor functions is associated with the complexity with which the hemispheres of the endbrain and cerebellum are organized. The functions of the latter include involvement in coordinating body movements and maintaining tone in the muscular system. Comparative studies of the cytoarchitectonics and cytometry of the cere-

Comparative Morphological Analysis of Neuron Populations

335

TABLE 1. Characteristics of Cerebellar Purkinje Cells in the Rodent Order (x ± sx) Parameter Cross-sectional area of body,

µm2

Cross-sectional area of cytoplasm, µm2 Cross-sectional area of nucleus,

µm2

Structural nuclear-cytoplasmic coefficient

House mouse

Gray rat

Mole lemming

Common vole

82.5 ± 1.1**

269 ± 4

193.1 ± 2.2**

175.9 ± 1.9**

43.7 ± 1.0**

191 ± 3

127.7 ± 1.7**

121.1 ± 1.6**

38.8 ± 0.7**

77.8 ± 1.6

65.4 ± 0.9**

55.1 ± 0.9**

0.98 ± 0.03**

0.40 ± 0.008

0.520 ± 0.007*

0.460 ± 0.009*

*p < 0.01; **p < 0.001 compared with gray rats.

TABLE 2. Numbers of Different Types of Purkinje Cells in the Cerebellar Cortex in the Rodent Order (x ± sx) Species

Normochromic

Hyperchromic

Hypochromic

Totally hyperchromic

Gray rat (Rattus norvegicus)

55 ± 4

41 ± 4

2.4 ± 0.9

0.96 ± 0.6

House mouse (Mus musculus L.)

71 ± 5*

26 ± 5*

2.6 ± 1.9

0.60 ± 0.20

Mole lemming (Ellobius talpinus P.)

56 ± 8

41 ± 8

1.8 ± 0.4

0.7 ± 0.3

Common vole (Microtus arvalis P.)

74 ± 4**

18 ± 5**

6.8 ± 2.8

0.6 ± 0.3

*p < 0.05; **p < 0.01 compared with Rattus norvegicus.

bella of rodents of different ecological-morphological groups allowed us to identify the common features in the organization and the specific characteristics of the neuron populations of interest. PC are the only efferent elements in the cerebellar cortex; they have inhibitory functions and are exclusively enriched with synaptic connections. These cells receive all spikes directed to the cerebellum, and all spikes from them pass through the cerebellar nuclei to the corresponding formations of the spinal cord and brain. In most rodents, PC were flask-shaped, with large, vesicle-like nuclei (Fig. 1); they formed straight ordered rows running along the gyrus. Within the extensive ecological-morphological group of small burrowing rodents, the species studied here differ not in terms of means of nutrition, daily activity, and mobility, but also in terms of the degree of synanthropy. The common vole and mole lemming are subterranean rodents, widely distributed in the open biotopes of steppe and forest regions, while the house mouse and the gray rat are constant companions of humans, burrowing animals with twilight activity, highly adapted to virtually all possible rodent habitats. Prolonged occupancy of certain ecological conditions is known [1, 7, 11] to lead to significant histological and cytological changes, both physiological and anatomical in nature, associated with interspecies differences at all levels of body organization. Our measurements of the distribution density of PC per mm gyrus length showed that animals of this ecological group can be listed in the following order of decreasing values of this parameter: common vole, house mouse, mole lemming, gray rat. The greater PC distribution densities

were seen in common voles and house mice (38.5 ± 1.8 and 37.1 ± 1.3 respectively (p < 0.001 compared with gray rats), followed by mole lemmings in an intermediate position (29.9 ± 0.8); gray rats had the smallest value (26.1 ± 1.3). Our data support the notion that the neuron distribution density changes not as a function of the species, but as a function of brain weight [1, 15]. Relative brain size is known to be smallest in rodents, while the neuron distribution density is highest in small animals; in this case this applies to the smallest burrowing mouse-type rodents – the common vole and the house mouse. Mean Sb values for PC in burrowing rodents were in the order: gray rat > mole lemming > common vole > house mouse. The extremes correspond to the mean values of Sc and Sn in synanthropic mouse-type rodents: the greatest values were seen in the gray rat and the smallest value was seen in the house mouse. Wild burrowing small rodents occupied an intermediate position (Table 1). Analysis of neuron populations showed that 69% of neurons in the PC population of the cerebellar cortex in gray rats had Sb from 200 to 300 µm2, Sc from 150 to 250 µm2 (79%), and Sn from 60 to 100 µm2 (67%). In wild burrowers, the PC population in common voles was dominated by cells with Sb from 140 to 200 µm2 (83%), while that of the mole lemming was dominated by cells with values of 160 to 220 µm2 (69%). Analogous data for Sc and Sn were: Sc varied from 100 to 160 µm2, the proportions of PC with these sizes being 70% of the population examined in common voles and 86% in mole lemmings; 50% of PC in common voles and 80% in mole lemmings had Sn from 40 to 80 µm2. Analysis of the data obtained here showed that gray rats and mole lemmings shared the following relationship:

336 the larger the PC, the lower their distribution density. While the PC distribution density per mm of gyrus in common voles and house mice were close, Sb for ganglion layer cells in house mice was 51.6% smaller than that in common voles. The PC population in house mice was characterized by the presence of small cells with Sb from 80 to 100 µm2, accounting for 54% of the population analyzed; these cells had large nuclei (35–45 µm2) and narrow rims of cytoplasm (cross-sectional area from 40 to 60 µm2); sNCC values for these cells were close to unity. This cardinal difference in cell size at identical distribution density may be associated with different extents of dendritic branching of PC and may be a type of adaptation to living conditions at the level of cerebellar neuron populations in wild and synanthropic animals. Morphologically similar PC are known to differ in terms of their staining properties; heterogeneity in a population correlates with difference levels of metabolism. Many authors hold that “dark” neurons represent the reserve capacity of a population, while “light” neurons are regarded as actively functioning cells [6, 9, 12]. Among the group of burrowing animals studied here, neuron populations in the ganglion layer showed differences in PC staining properties (Table 2). The largest proportions of normochromic neurons were found in house mice and common voles. In mole lemmings and gray rats, the numbers of normochromic cells was only half the population and were the lowest among the cell populations studied here. The opposite relationship was seen for “dark,” i.e., hyperchromic neurons: the greatest numbers were seen in the PC populations of mole lemmings and gray rats, while common voles had the smallest number. Light neurons, as compared with dark cells, were less typical of the ganglion layer of the cerebellum in rodents; they were more frequently seen among burrowers and were virtually absent from the PC population of mole lemmings. Totally hyperchromic neurons were more typical for the PC population of gray rats. It should be noted that most neurons in the PC populations of mole lemmings and gray rats accumulated RNP complexes, these not extending beyond the limits of the cell. These cells were in a state of reserve for the neuron population, playing an identifiable role in the rapid redistribution of functional loading between neurons within cell populations; this is a component required for maintaining a relatively constant level of interaction between the body and its environment. The differences in the linear sizes of PC, their distribution densities per unit gyrus length, and their staining heterogeneity in burrowing rodents allow the data obtained

Orlyanskaya and Lyutikova here to be regarded as a type of idioadaptation of the ecological-morphological group of mammals studied at the cell population level, which explains their morphofunctional variability.

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